It is known that many proteins are modified in various manners after being translated. Among the modifications, phosphorylation of proteins is important as a factor for changing various physiological activities and enzymatic activities of proteins to regulate intracellular signal transduction or intracellular metabolic activities. Therefore, it is very important to analyze intracellular phosphorylation of proteins. Conventionally, various techniques have been developed for examining phosphorylation of proteins, one of which uses mass spectrometry1).
Mass spectrometry is practically performed as follows. Proteins separated and purified by electrophoresis or the like are treated with enzymatic digestion, and measured by MALDI-TOF/MS or the like. The resultant peptide-mass fingerprint is checked against a database to identify proteins. When there is a peptide chain which is larger by 80 Da than the theoretical value obtained from the primary amino acid sequence, there is a high possibility that one site of the peptide chain is phosphorylated. Then, the peptide chain is treated with alkaline phosphatase to specifically remove the phosphoric acid group from the peptide chain, and the peptide chain is again measured by mass spectrometry. When the peptide chain is now smaller by 80 Da and matches the theoretical value, it is proved that the peptide chain was phosphorylated at one site2).
Regarding phosphorylation of one protein, it is possible to identify the phosphorylated peptide and the phosphorylated site by digesting the proteins and analyzing peptide fractions using a mass spectrometer.
In proteome analysis, however, several hundred to several thousand proteins are measured at once. The term “proteome analysis” refers to an analysis of clarifying the relationship between the gene information and various types of proteins interacting in a cell in a complicated manner3). Namely, proteome analysis refers to a technique of comprehensively analyzing all the proteins included in the cell.
For this reason, in proteome analysis, it is extremely difficult to check mass spectra one by one. In most cases, the result provided by an automatic search engine (e.g., MASCOT) is accepted as being correct with no checking.
Usually, proteome analysis uses a protein database (e.g., NCBInr, IPI, or Sport). When an automatic search engine (e.g., MASCOT) is used, a great number of pseudopositive and pseudonegative results are provided and the search requires a huge amount of time. For these reasons, it is difficult to perform proteome analysis with high efficiency and high precision.
Usually, most proteins contain phosphorylated molecules and non-phosphorylated molecules in a mixed state. There is almost no protein in which most protein molecules are phosphorylated. One protein molecule is occasionally phosphorylated at a plurality of sites, or is mixed with other types of proteins. Therefore, it is difficult to directly detect a phosphorylated protein by mass spectrometry.
In addition, it is generally known that when a protein is phosphorylated, the detection sensitivity for the protein by a mass spectrometry is decreased. Therefore, when the intended proteins are not purified to a high degree or are only purified in a small amount, it is difficult to detect phosphorylated proteins. When the amount of proteins in a sample is extremely small, it is very difficult to detect phosphorylated proteins. In order to comprehensively analyze phosphorylated proteins, it is desirable to first specifically purify and then measure the phosphorylated proteins with a mass spectrometer.
One generally used method for specifically purifying phosphorylated proteins is immobilized metal affinity chromatography (hereinafter, occasionally referred to as “IMAC”). An IMAC column is formed of metal such as tertiary iron ions or gallium immobilized on a chelate forming group with a plurality of carboxylic acids. Since a phosphoric acid group specifically and strongly binds to tertiary iron ions, a phosphorylated protein can be bound to the IMAC column. For binding a phosphoric acid group to the IMAC column, acid conditions are used. For releasing the phosphoric acid group, the pH value of the solvent is made weakly alkaline or competitive elution means using a phosphoric acid buffer solution is used4-12).
However, it is not easy to purify only phosphorylated proteins using the IMAC column for the following reason. Because the carboxylic acids have affinity to the IMAC column, peptides having acidic amino acids are more or less bound to the IMAC column.
In order to solve this problem, a purification method of digesting the proteins with trypsin and then methylesterifying the carboxylic acids in a methanol anhydride-hydrochloric acid solution so as to suppress the adsorption of the acidic amino acids to the IMAC column has been reported13). With this method, however, the phosphorylated proteins are, often times, not specifically purified for the following reasons14). The esterification does not quantitatively proceed, side effects occur, the selectivity is not improved as expected, or the peptides are insolubilized after the esterification.
In addition, dihydroxy benzoic acid (hereinafter, occasionally referred to simply as “DHB”), which is conventionally used for purifying phosphorylated proteins, is usable for MALDI-MS, but is not usable for LC-MS.
Under such a situation, it is difficult to detect phosphorylated proteins.